The objective of this work is to characterize in mechanistic detail the way by which certain hydrolytic enzymes function. Information obtained from these studies could contribute to the development of improved methods for treating and characterizing certain metabolic diseases in man which arise from an abnormality in a hydrolytic enzyme. Interactive effects of ionizable groups at the active site of papain on the ionization of the active site thiol group will be investigated by pH-difference titrations of derivatives of papain relative to papain. The ionization of the histidyl residue at the active site of papain will be studied by means of proton-nmr titrations. Individual steps in the catalytic pathway will be examined using 13C-enriched dinitrophenyl peptides by means of 13C-nmr and stopped-flow spectrometry. Individual steps in the catalytic pathway for the D-serine dehydratase-catalyzed reaction will be characterized by studying the presteady-state, and kinetic isotope effects on the presteady-state. The facilitation of amide hydrolysis by hydrogen bonding at the carbonyl oxygen of the scissile bond will be studied as a model for the action of certain hydrolytic enzymes. BIBLIOGRAPHIC REFERENCES: Lewis, S.D., Johnson, F.A., and Shafer, J.A. (1976) Potentiometric Determination of Ionizations at the Active Site of Papain, Biochemistry 15, 5009-5017. Lewis, S.D., Johnson, F.A., and Shafer, J.A. (1976) Potentiometric Determination of the Ionization of Groups at the Active Site of Papain, Amer. Chem. Soc. Biol. Chem. Div. 41, Abs.